Cervical cancer is the second most common cause of cancer-related deaths in women worldwide. There is both epidemiological and experimental data which links the etiology of cervical cancer to infection with human papilloma virus (HPV) types 16 and 18. The HPV virus is prevalent in 35 to 40% of young women. Although treatment of early stage disease is relatively successful, recurrent disease is found in 15% of the patients. The outcomes of patients with recurrent disease are relatively poor. Hence, there is a need for a novel therapeutic approach (refs. 1, 2, 3--various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosure of these references are hereby incorporated by reference into the present disclosure).
The strong association of HPV infection and cervical cancer suggests that a viral antigen-specific immunotherapeutic approach may be a feasible strategy in the treatment of cervical cancer. The goal of specific immunotherapy is to stimulate the immune response of a tumour-bearing patient to attack and eradicate tumour lesions. This strategy has been made feasible with the identification of tumour associated antigens (TAA). The strong association between HPV-16 infection and cervical cancer has made this disease a good candidate for immunotherapeutic intervention (ref. 4).
In HPV DNA-positive cervical cancers, the E6 and E7 oncogenic proteins are expressed. Experimental evidence suggests that these two proteins are responsible for the carcinogenic progression of cervical cancers as their expression leads to a transformed and immortalized state in human epithelial cell cultures (ref. 5). Therefore, these two proteins are potential candidates for antigen-specific immunotherapy in HPV-induced cervical cancers and are evaluated herein.
Although many questions remain regarding the nature of immunity to natural HPV infection, and, in turn, to cervical cancer, it is clear that there is an immune component as immunosuppressed individuals are at higher risk for developing a cervical malignancy (ref. 6). Furthermore, this immunity is most likely mediated by the cellular arm of the immune response. Extensive cellular infiltrates are observed upon examination of spontaneous regressions of cervical tumours (ref. 7). Thus, an antigen-specific cellular response appears to be required to treat cervical cancer patients.
Although the nature of the outcome of an immunotherapeutic strategy has been identified, the ability to induce this type of response using current vaccine technology is limited. Prophylactic vaccine development for HPV has focussed on recombinant subunit preparations consisting of L1 and L2 virion structural proteins. In eukaryotic cells, L1 (major capsid protein) organizes itself into papillomavirus-like particles (VLPS) (ref. 17). Although L1 alone is sufficient for assembly of VLPs, the coexpression of L2 (minor capsid protein) provides for greater capsid production (ref. 18). By contrast, therapeutic vaccine development has typically been directed to the expression of wild type E6 and/or E7 protein. Expression vectors employed include vaccinia virus (for example, as described in U.S. Pat. No. 5,744,133, (ref. 19, 20), alphavirus (for example U.S. Pat. No. 5,843,723), or other poxviruses (for example, U.S. Pat. No. 5,676,950). Therefore, a DNA vector encoding HPV antigens implicated in carcinogenic progression of disease was determined to be the optimal method by which a successful immunotherapeutic strategy could be achieved.
However, as previously noted the E6 and E7 HPV antigens are putatively oncogenic and thus immunization with a DNA construct encoding either or both of these proteins could result in the induction of further malignancy (refs. 5, 10, 11). Therefore, in order to minimize toxicity risks, a genetically detoxified E7 molecule was encoded herein in a DNA construct. This detoxified molecule is modified through the deletion of the retinoblastoma (Rb) binding region (refs. 8, 9). Another method of achieving antigen-specific immunity without the concomitant risks of oncogenic transformation is the use of an epitope strategy where only key parts of the molecule are administered to induce a specific immune response (refs. 12 to 16). This approach was used herein in the design of the DNA-polyepitope construct where a number of T-cell epitopes derived from both E6 and E7 are linked together. A comparison of these two approaches was made herein in a murine model of HPV-associated cervical cancer.